The long-term goal of this project is to develop an effective and rapid in vitro diagnostic (IVD) test for the evaluation of patient vaginal fluid specimens for vulvovaginal candidiasis using secreted aspartyl proteinase (SAP) as a biomarker. Vulvovaginal candidiasis, the second leading form of infectious vaginitis, and the most common reason cited by women for telephoning or visiting their OB/GYN physician, is traditionally self-diagnosed by patients or diagnosed during telephone triage by a nurse based on patient answers to questions posed by the nurse and a review of patient history. Less commonly, clinicians are involved in diagnosis of patients for vulvovaginal candidiasis based on microscopic methods and a review of symptoms. The most common antifungal treatments are over-the-counter fluconazoles or clotrimazoles. Numerous studies demonstrate very poor correlation between the answers provided by patients to questions posed during telephone triage and answers provided to clinicians during a clinical visit, very poor correlation between diagnosis by nurses during telephone triage and clinicians during a near immediate office visit, and the inaccuracy of current clinical methods of diagnosing vulvovaginal candidiasis. An inexpensive, reliable, and efficient IVD for vulvovaginal candidiasis of high clinical performance would be readily welcomed by both the clinical community and patients, greatly assisting physicians and/or patients in confidently and rapidly charting a course of treatment, and significantly reducing the clinical manifestations, increased medical costs, and increased office visits associated with empiric or incorrect diagnosis. Previous work has linked SAP with vulvovaginal candidiasis, the pathogen most associated with infection (i.e., C. albicans), and demonstrated significantly higher levels of SAP in women with vulvovaginal candidiasis compared to carriers. Significant work has been conducted to establish characteristics of the enzyme and its isoenzymes. Our specific aims draw largely on these previous studies and include: (a) synthesis of suitable colorimetric substrates of SAP2 (both chromogenic substrates and a trapped enzyme reported target) and (b) evaluation of substrates with purified SAP2 demonstrating their commercial utility in the development of an IVD for vulvovaginal candidiasis. [unreadable] [unreadable]